The effect of the cellulose-binding domain from Clostridium cellulovorans on the supramolecular structure of cellulose fibers

Carbohydrate Research 345 (2010) 621–630

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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres

The effect of the cellulose-binding domain from Clostridium cellulovorans on the supramolecular structure of cellulose fibers Diana Ciolacu a,b, Janez Kovac c, Vanja Kokol a,* a

Department of Textile Materials and Design, Institute of Engineering Materials and Design, University of Maribor, Smetanova ulica 17, 2000 Maribor, Slovenia ‘Petru Poni’ Institute of Macromolecular Chemistry, 41A Grigore Ghica Voda Alley, 700487 Iasi, Romania c Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia b

a r t i c l e

i n f o

Article history: Received 21 September 2009 Received in revised form 14 December 2009 Accepted 22 December 2009 Available online 29 December 2009 Keywords: Cellulose-binding domain Cellulose supramolecular structure FTIR XRD XPS SEM

a b s t r a c t The cellulose-binding domain (CBD) is the second important and the most wide-spread element of cellulase structure involved in cellulose transformation with a great structural diversity and a range of adsorption behavior toward different types of cellulosic materials. The effect of the CBD from Clostridium cellulovorans on the supramolecular structure of three different sources of cellulose (cotton cellulose, spruce dissolving pulp, and cellulose linters) was studied. Fourier-transform infrared spectroscopy (FTIR) was used to record amides I and II absorption bands of cotton cellulose treated with CBD. Structural changes as weakening and splitting of the hydrogen bonds within the cellulose chains after CBD adsorption were observed. The decrease of relative crystallinity index of the treated celluloses was confirmed by FTIR spectroscopy and X-ray diffraction (XRD). X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used to confirm the binding of the CBD on the cellulose surface and the changing of the cellulose morphology. Ó 2010 Published by Elsevier Ltd.

1. Introduction The enzymatic hydrolysis of cellulose is a complex heterogeneous reaction with a fundamental initial step comprising the adsorption of cellulases to the cellulose surface. Most of the cellulolytic enzymes include a modular multi-domain protein containing at least three structural elements of different functions, that is, a catalytic domain (CD), a cellulose-binding domain (CBD), and inter-domain linker, among which the CBD is the second important and the most widespread element involved in the cellulose transformation.1,2 According to the amino acid sequences, binding specificity, and structures, the carbohydrate-binding modules (CBMs) are divided into more than 40 different families, and the CBDs are distributed in the first 13.3,4 Extensive data can be found in the Carbohydrate-Binding Module Family Server (http:// www.afmb.univ-mrs.fr/CAZY). The CBDs range in the size from approximately 33–36 amino acids in family I to 180 amino acids in family III. Even if the amino acid sequence within a family is highly conserved, a significant difference has been found between several of these CBDs. The first evidence that CBMs have potential in modification of cellulosic fiber was demonstrated by their non-hydrolytic fiber disruption activity. It was proposed that these treatments could be used to alter the dyeing characteristics of cellulose fibers.5 Addi* Corresponding author. Tel.: +386 2 220 7896; fax: +386 2 220 7990. E-mail address: [email protected] (V. Kokol). 0008-6215/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.carres.2009.12.023

tional information was reported by Cavaco-Paulo et al.6 who demonstrated elevated levels of dye affinity of cotton cellulose following treatment with family II CBMs from Cellumonas fimi. There are still controversies on the interaction mode of CBD with cellulose. CBDs from the same organism can differ in their binding specificities, and occasionally two CBDs located on the same enzyme can also exhibit this distinction. Thus, Carrard and Linder7 report that the binding is a totally reversible process for CBHI–CBD from Trichoderma reesei, while it is irreversible for CBDs from C. fimi according to Brun et al.,8 who confirmed that the CBDCex (the gene fragment encoding the CBD of an exoglucanase) is mobile on the surface of the crystalline cellulose. The strong affinity that exists between cellulose and CBD can be used in many applications, a fact that demonstrates the technological significance of it. The first commercial potential of CBDs in the textile area was realized for denim stonewashing, where cellulases were used as an alternative to the original abrasive stones, and the presence of CBD allowed for the targeting of the enzyme onto the garment.9 Another application is related with laundry powder, which contains recombinant enzymes without a native affinity to the cellulosic fabric (e.g., amylase, protease, and lipase). It was shown that the performance of these enzymes can be improved by fusion to CBDs.10 Fragrance-bearing particles, conjugated with CBD, can be added to the laundry powder, hence reducing the amount of fragrance needed in the product.11 In addition, antimicrobial agents (aromatic aldehydes or alcohols, e.g., acetaldehyde, cinnamaldehyde, and vanillin) can be targeted to polysaccharide

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materials. Moreover, there are studies related to the significant improvement of tensile strength of filter paper treated with CBD or CCP (cross-linked protein),4 while cellulose fibers treated with CBD have a potential for use in paper recycling.12 It becomes obvious that it is important to understand the structural modifications of cellulose-based fibers following the adsorption of CBDs. Our efforts to understand the nature of the CBD–cellulose interactions have been focused on the binding capacity of cellulose and on extensive structural changes of different cellulose fibers after their treatment with CBD from Clostridium cellulovorans. To establish the supramolecular modification, which takes place at the hydrogen-bonding level within a specific cellulosic fiber after CBD adsorption, Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) were used. In addition, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were applied to gather more information about the adsorption process at the surface of a cellulose substrate. 2. Experimental 2.1. Materials Three types of cellulose were used through the study: cotton cellulose (B; Arshad Enterprises, Pakistan), spruce dissolving pulp (C; Extranier F, Rayonier, France), and cotton-linter cellulose (L; Sigma–Aldrich, Germany). The cotton-linter cellulose (L) and spruce dissolving pulp (C) were used without any purification, while the cotton cellulose (B) was extracted for 8 h in a Soxhlet extractor using ethanol and benzene, after which it was boiled in 1% NaOH for 6 h, washed with distilled water, immersed in 1% acetic acid, washed with water, and finally air dried. CBD from C. cellulovorans, expressed in Escherichia coli, was purchased from Sigma–Aldrich and by SDS–PAGE the molecular weight was 17 kDa and the purity was >95%. All other chemicals used were of the highest purity available commercially. 2.2. CBD adsorption studies A suspension of cotton cellulose fibers with a concentration of 1 mg per mL of phosphate buffer (100 mM, pH 7) was incubated in the presence of different CBD concentrations (4–22 lg CBD per 1 mg of fibers) for 2 h at 35 °C with gentle rotation (400 rpm). In parallel, blank samples were prepared similarly, but without CBD. Afterward, the fibers were centrifuged at 5000 rpm for 10 min, and the CBD concentration in the supernatant (PF, lM) was measured in a TECAN Infinite M200 spectrophotometer. The protein concentration was determined by the Lowry method using the Bio-Rad DC Protein assay. The bound CBD (PB, lM) was calculated using the following equation:

PB ¼ ½ðPT  P F Þ=m  V ðlmol=gÞ

ð1Þ

where PT is the initial concentration of CBD, lM; PF is the free CBD concentration, lM; PB is the bound CBD concentration, lM; m is the mass of the cellulose fibers (g); and V is the volume of the buffer (L). The fibers were washed with the same volume of buffer, centrifuged at 5000 rpm for 5 min, and lyophilized using Mini Lyotrap freeze-dryer. The protein adsorption on cellulose was studied by the Langmuir isotherm, which assumes that the adsorption can be described by single adsorption equilibrium constant and a specified adsorption capacity.13

PB ¼

PMax  K d  P F 1 þ K d  PF

ð2Þ

where PMax is the maximum protein adsorption, lmol CBD/g; and Kd is the dissociation constant, L/lmol.

The data were analyzed by non-linear regression analysis of 1/PB versus 1/PF. The distribution coefficient, R, was defined as

R ¼ K d  PMax

ð3Þ

where R is the dimension, L/g cellulose. Average values of three measurements (replicates of adsorption studies) are given as the final results. 2.3. ATR–FTIR analysis ATR–FTIR spectra of all samples were recorded using a Perkin– Elmer Spectrum One GX FTIR spectrometer with a Golden Gate ATR attachment and diamond crystal. The absorbance measurements were carried out in the range of 400–4000 cm1, with 16 scans and a resolution of 4 cm1. 2.4. X-ray diffraction method X-ray diffraction patterns of the samples were collected on a Bruker D8 Advance apparatus, equipped with a transmission-type goniometer using nickel-filtered, Cu Ka radiation. The resulting diffraction patterns exhibited peaks that were deconvoluted from a background scattering by using Lorenzian functions, while the diffraction pattern of an artificially amorphicized sample was approximated by a Gaussian function curve-fitting analysis. The values of the crystallinity index of cellulose samples were determined by the surface method (CrI1) which corresponds to the ratio between crystallinity area (SC) and total area (ST), and by the Segal method (CrI2) which is estimated from the intensity of the peaks corresponding to crystalline and amorphous areas:14

SC  100ð%Þ ST I002  Iam  100ð%Þ Cr  I2 ¼ I002 Cr  I1 ¼

ð4Þ ð5Þ

The average size of the crystallites, measured in the directions  and (0 0 2) planes, was calculated orthogonal to the (1 0 1), (1 0 1), 15 with the Scherrer equation:

DðhklÞ ¼

Kk bðhklÞ  cos h

ð6Þ

where D(hkl) is the average crystallite size, k is the wavelength of the incident X-ray (1.5418 Å), b(hkl) is the FWHM (full width at half maximum) of the reflex in radial direction, h is the Bragg angle corresponding to the crystallographic planes, and K is the Scherrer constant (0.89). 2.5. XPS analysis The XPS analysis, also known as Electron Spectroscopy for Chemical Analysis (ESCA), was carried out on a PHI-TFA XPS spectrophotometer produced by Physical Electronic Inc. The untreated and variously treated cellulose fibers were fixed by using a metallic plate with a hole. Thereby, three different sample locations were analyzed from two experimental series. The analyzed area was 0.4 mm in diameter, and the analyzed depth was 3–6 nm on the cellulose samples. The surfaces of the samples were excited by X-ray radiation from a monochromatic Al source at a photon energy of 1486.6 eV. Widescan spectra were acquired at a pass energy of 187 eV for identification and quantification of elements on the fiber surfaces. In order to reveal binding energies of XPS peaks associated with different chemical states of elements, the narrow-scan spectra C 1s, O 1s, and N 1s were acquired with an energy resolution of about 0.65 eV measured at the Ag 3d5/2 spectrum. A low-energy electron gun—neutralizer was used due to

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sample charging. In addition, during data processing the XPS spectra were aligned by setting the C–C/C–H peak in the C 1s spectrum to a binding energy of 285.0 eV.16 The accuracy of binding energies was about ±0.3 eV. Concentrations were calculated from intensities of the peaks in the XPS spectra using relative sensitivity factors provided by the instrument manufacturer.16 On each sample two spots were analyzed, and the average composition was calculated. The composition was calculated assuming homogenous elemental distribution throughout the analyzed volume. 2.6. SEM analysis The morphology of the cellulosic samples and the presence of the CBD on the fibers surface were studied by SEM on a low vacuum scanning electron microscope, a FEI QUANTA 200 3D instrument. The fibers were coated with gold particles before examination.

Figure 2. The non-linear regression of CBD binding to the cotton cellulose (B), linters cellulose (L), and dissolving pulp (C).

3. Results and discussion 3.1. CBD adsorption on different cellulose fibers The adsorption isotherms for all three cellulose fibers at 35 °C, obtained after 2 h using CBD solutions with different concentrations, are presented in Figure 1. The differences in the adsorption process can be observed. Thus, for cotton cellulose (B) with the highest concentration of bound CBD, the rate of CBD adsorption was much higher than those for linters cellulose (L) and dissolving pulp (C). The differences in rates are explained by the differences in specific surface areas and the crystalline structures of the samples. The linearity of diagnostic plots of the CBD binding to each cellulosic substrata, presented in Figure 2, describes the partitioning of the CBD between the solid and liquid phase at low surface coverage, where lateral interaction between adsorbed proteins do not influence the binding. The highest Langmuir adsorption parameters (Table 1) are recorded for cotton cellulose, and they decrease with linters cotton (L) and dissolving pulp (C). Generally, the maximum protein adsorption, PMax, and the distribution coefficient, R, have been selected as the index of binding capacity, while the dissociation constant, Kd, is an index for estimating the affinity.17 Thus, a smaller value of the constant means a greater affinity of CBD toward the substratum. The data obtained show that the maximum molar amounts of absorbed protein, PMax, for cotton cellulose (2.4 lmol/g) and cotton-linters cellulose (2 lmol/g) are intermediate values between

Table 1 Langmuir cellulose adsorption parameters for CBD Sample

PMax (lmol/g)

Kd (L/lmol)

R (L/g)

B—cotton cellulose L—linters cellulose C—dissolving pulp

2.361 2.066 1.677

1.954 2.867 3.171

4.615 5.924 5.316

those reported in the literature,13 which indicated a value of 6.4 lmol/g for highly crystalline cotton and 0.2 lmol/g for fibrous cotton that was less crystalline in form. These results could be explained by the differences in the experimental conditions and in the crystallinity index of the samples, which appears to be a key parameter in CBD-binding capacity.1,18 The smaller Kd value for cotton cellulose reflects the highest potential for protein interaction sites of this sample. The affinity of CBD toward the substratum decreases in the L and C samples. Based on the literature data, the crystallite length for cotton linters is reported to be 8.5–10 nm, and for dissolving pulp, 7.5– 9.7 nm.19 In the present study the CBD from C. cellulovorans, expressed in E. coli, subfamily IIIa, with a length of 16.1 nm was used (http://www.afmb.univ-mrs.fr/CAZY).1 Thus, the dimensions of the CBD used exceed those of the repeating cellobiose lattice units exposed on the surface of cotton cellulose microfibrils. Based on these facts it can be assumed that the adsorption involves interaction with more than one lattice unit. 3.2. X-ray diffraction measurements

Figure 1. The adsorption isotherms of CBD on the cotton cellulose (B), linters cellulose (L), and dissolving pulp (C).

The cellulose crystalline and supramolecular structures are one of the most important parameters that have to be taken into account for the understanding of CBD adsorption to cellulose. In order to observe the structural modifications of cellulosic samples during protein adsorption, the deconvolution of the peaks was performed using the soft PeakFit 4.11. In Figure 3 are presented the X-ray diffractograms of selected celluloses with the characteristic shapes of cellulose I crystalline structure and corre and (0 0 2) which sponding crystallographic planes (1 0 1), (1 0 1), appear at a Bragg angle of 14°, 16°, and 22°, respectively. The slight decrease of the intensity of corresponding peaks for untreated cellulose samples (from B via L to C) confirms the decrease in degree of crystallinity between them. The influence of the adsorption process was studied only for cotton cellulose samples presenting the highest value for the maximum protein adsorption. In Figure 4, the structural modifications of cotton cellulose treated with the lowest (B1) and the highest

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Figure 3. X-ray diffractograms of the cellulosic samples (a) B sample; (b) L sample; (c) C sample.

(B6) concentrations of CBD are shown, and it is observed that an important decrease of the peak corresponding to the (0 0 2) plane which appears at a Bragg angle of 22° during CBD adsorption pro becomes sharper and higher in cess. Also, the reflection from (1 0 1) comparison with that from the (1 0 1) plane, which remains invariable. These results indicate that CBD preferentially adsorbs to the (0 0 2) crystallographic planes from the crystalline form of cotton cellulose I. As presented in Table 2, a slight decrease in the crystallinity index of the cellulose samples is observed during the CBD adsorption process (from samples B1 to B6). This fact could be explained by an adsorption of CBD at the cellulose surface, followed by a slight penetration of CBD between the chains, which consequently results in a less dense and less ordered structure. The reduction in the degree of crystallinity is in good agreement with that reported by others who studied the structural changes of cellulose after treatment with CBD of endoglucanase III (CBDEG III) from T. reesei.20It can be observed that the dimensions of the crystallite sizes in the (0 0 2) directions decrease with the increase of

Table 2 Degree of crystallinity and crystallite dimensions of cellulosic samples Sample

C L B B1 B6

Degree of crystallinity (%)

D(hkl) (nm)

CrI1

CrI2

(1 0 1)

 (1 0 1)

(0 0 2)

65.47 71.11 80.07 78.75 75.15

75.45 83.33 86.21 82.71 80.99

6.63 7.41 7.87 8.12 8.68

5.79 6.70 7.59 8.49 9.08

7.89 9.35 9.02 8.42 7.01

 direction increases from the the concentration of CBD. The (1 0 1) untreated cellulose to the sample treated with the highest concentration of CBD, while the (1 0 1) direction remains approximately constant, being recorded as just a slight increase. From the data obtained it can be observed that CBD may have different affinities for different crystal faces of crystalline cellulose, but it shows preference for the (0 0 2) plane. The proposed mechanism of binding to cellulose shows the attachment of CBD to the (0 0 2) crystallo-

Figure 4. X-ray diffractograms of cotton cellulose treated with (a) the lowest (B1) and (b) the highest (B6) concentrations of CBD.

D. Ciolacu et al. / Carbohydrate Research 345 (2010) 621–630

Figure 5. FTIR spectra of CBD, cotton cellulose (B), and cotton cellulose treated with CBD (B6).

graphic face that, probably due to their hydrophobic character, would only be exposed at the edge of cellulose crystals. There is experimental evidence that shows that CBD penetrates the fibers and preferentially binds to the edge of the cellulose microfibrils.21,22 It can be concluded that the changes in lattice dimensions and chain conformation during the CBD adsorption process first appear in the amorphous domain, and then the supramolecular structural regularity of the ordered phase of cellulose is affected. The explanation could be that the adsorption of CBD causes a slight disruption of intermolecular association between the parallel chains, and together with water molecules releases the chains from the crystal. 3.3. ATR–FTIR measurements The hydrogen-bonding patterns in cellulose are considered as one of the most influential factors in determining the structure

625

and properties of cellulosic materials. In order to establish the modification that takes place at the level of the hydrogen bonds in the cellulosic structure after it is treated with CBD, FTIR analysis of the cotton cellulose with the highest protein adsorption was carried out. For a systematic and rigorous study, all the spectra will be discussed regarding the most important FTIR bands. The changes of many FTIR bands characteristic of cotton cellulose reflect the occurrence of the CBD adsorption onto cellulose (Fig. 5). The intensity of hydrogen bonds in region of 3600–3200 cm1 is significantly decreased, and those of the amide I (1700–1600 cm1) and amide II (1600–1500 cm1) regions increased during the CBD adsorption process. The ‘fingerprint region’ of 1400–900 cm1 exhibits a few changes in the position of the absorption bands and a slight decrease in their intensity. The broad band between 3700 and 3000 cm1, which is due to the OH-stretching vibration, gives considerable information concerning the hydrogen bonds. The assignments for intramolecular hydrogen bonds of O(2)H  O(6) and O(3)H  O(5), and intermolecular hydrogen bonding of O(6)H  O(3) in the cellulose crystalline structure are generally shown at 3410–3460 cm1, 3340–3375 cm1, and 3230– 3310 cm1, respectively.23 Since the characteristic absorption peaks of cotton celluloses in the range between 3600 and 3100 cm1 were broad and overlapped (spectra not shown), the resolution of the spectra was improved by their deconvolution from a background scattering using a Gaussian function curve-fitting analysis (Fig. 6). It is observed that the intensity of the OH-stretching vibration decreases with the binding of CBD, which can be correlated with a disruption of intra- and intermolecular hydrogen bonds (Table 3). The more evident changes take place at the intermolecular hydrogen bonds, whose intensities decrease about 23% for the sample treated with the highest CBD concentration compared to the blank sample. The intensities of the intramolecular hydrogen bonds established between O(3)H  O(5) appear to be more affected by the adsorption of CBD (21%).Pimentel and Sederholm have found linear relationships between the bond distance (R) for O–H  O and the frequency shift (Dm) of the OH absorption band caused by hydrogen bonding, and expressed it as follows:24

Dm ¼ 4:43  103 ð2:84  RÞ

ð7Þ

Figure 6. Deconvoluted FTIR spectra in (a) the range between 3300 and 3600 cm1 for cotton cellulose, (b) blank cotton cellulose, and (c, d) cotton celluloses treated with CBD concentrations.

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Table 3 FTIR absorption bands’ assignment to the OH range (3600–3000 cm1) of cotton cellulose (B) and cotton cellulose after its treatment with CBD (B1–B6) Sample

Intermolecular hydrogen bonds of O(6)H  O(3) 1

B BK B1 B2 B3 B4 B5 B6

Intramolecular hydrogen bonds of O(3)H  O(5) 1

cm

Intensity

Modif. (%)

cm

Intensity

Modif. (%)

cm1

Intensity

Modif. (%)

3279.63 3275.84 3275.26 3272.32 3272.35 3271.99 3272.32 3272.32

0.128 0.127 0.126 0.125 0.121 0.118 0.106 0.103

— — 0.79 1.60 4.96 7.63 19.81 23.30

3362.56 3366.05 3366.21 3366.21 3366.21 3369.56 3372.32 3372.45

0.116 0.116 0.114 0.113 0.114 0.107 0.097 0.096

— — 1.75 2.65 1.75 8.41 19.59 20.83

3476.90 3480.40 3483.58 3483.58 3483.58 3483.58 3486.93 3487.16

0.064 0.065 0.062 0.059 0.059 0.058 0.059 0.054

— — 1.64 5.08 5.08 6.90 5.08 14.81

Generally, it was accepted that in the structure of native cellulose, intramolecular hydrogen bonds of types O(3)H  O(5) and O(2)H  O(6) are present on both sides of the chain. The bond length of O(3)H  O(5) is reported to be 0.275 nm, and the length of the O(2)H  O(6) hydrogen bond is reported to be 0.287 nm. An intermolecular hydrogen bond, O(6)H  O(3), is formed with a length estimated to be 0.279 nm. It is known and accepted that these hydrogen bonds play an important role in determining the conformational and mechanical properties of cellulosic materials.23,25 In order to describe the absorption of CBD onto the cellulose structure, the length of the hydrogen bonds (R), the energy of the hydrogen bonds (EH), and the index of asymmetry (a/b) were determined on all samples studied. The energy of the H-bonds was calculated from the equation:26

EH ¼

Intramolecular hydrogen bonds of O(2)H  O(6)

1 ðm0  mÞ x k m0

ð8Þ

where mo is the standard frequency corresponding to free –OH groups (3600 cm1), m is the frequency of the bonded –OH groups, and k = 1.68  102 kcal1. The asymmetric index (a/b) was determined from the ratio between segment widths at half height of the OH absorption band.27 As can be observed from the results collected in Table 4, the hydrogen-bond (R) values obtained for all celluloses treated with CBD are higher than the ones reported for sample B, a fact that provides evidence that the hard-segment domains are packed in a less ordered arrangement. (Note that shorter hydrogen bonds are generally stronger.) The highest energy of the hydrogen bonds (EH) was recorded for sample B, and this value decreases after CBD binding, indicating a decrease of the number of hydrogen bonds and consequently changes in the crystalline structure of cellulose, that is, a decrease in the degree of crystallinity. The index of asymmetry (a/b), which characterizes the uniformity of cellulosic samples, indicates that the most uniform samples are those of cotton cellulose, and the less uniform samples arise from cellulose treated with the highest concentrations of CBD. This parameter shows that the CBD is randomly absorbed lengthwise along the cellulosic fiber. It can be concluded that the interaction occurred between the cel-

lulose and CBD, resulting in conformational changes of the cotton cellulose structure. The realization of CBD binding to cotton cellulose can be further confirmed by the shifting of the band at 2900 cm1 (Fig. 5), corresponding to the C–H stretching vibration, from 2899 cm1, the absorption band of cotton cellulose, to 2904 cm1 characteristic for cellulose treated with the highest concentration of CBD (B6). In addition, the intensity of this band decreases, and the shape of it seems to be more like that of CBD at 2972 cm1. Although the hydrogen-bonded N–H stretching vibration of the amide (amide A), protein-specific band, appears in the range of 3300–3400 cm1, it is difficult to identify them because of their overlap with the band that is characteristic of the OH-stretching vibration of the cellulose crystalline structure.28 However, the polypeptide and protein repeat units give rise to nine characteristic FTIR absorption bands, namely, amide A, B, and IVII, among which the amide I and II bands are two the most prominent vibrational bands of the protein backbone.29 The amide I absorption band is located in the region between 1700 and 1600 cm1, which is the most sensitive spectral region for the identification of protein secondary structural components,30 due almost entirely to the C@O stretch vibrations of peptide linkages coupled with in-plane N–H bending and C–N stretching. The position of this band in that region indicates the conformation of the protein in terms of a-helices, b-sheets, turns, and random-coil structures.31,32 The amide II band, which falls in the 1600–1500 cm 1 range, derives mainly from in-plane N–H bending (4060% of the potential energy) and from the C–N stretching vibration (1840%), showing much less protein conformational sensitivity than amide I. Other amide vibrational bands are very complex, depending on the details of the force field, the nature of side chains, and hydrogen bonding, all of which are of little practical use in protein conformational studies. The FTIR spectrum of CBD shows a peak at 1636 cm1 for amide I and a peak at 1522 cm1 for amide II (Fig. 5), indicating the secondary structure of the protein consists mainly of b-sheet and random-coil conformations.33,34 The modifications that take place at the protein level during the adsorption process are recorded as a shifting of the characteristic amide peaks to higher wavenumbers.

Table 4 The length of intra- and intermolecular hydrogen bonds in cotton cellulose (B) before and after its treatment with different concentrations of CBD (samples B1–B6) Sample

B BK B1 B2 B3 B4 B5 B6

Intramolecular hydrogen bonds of O(3)H  O(5)

Intramolecular hydrogen bonds of O(2)H  O(6)

R (nm)

Intermolecular hydrogen bonds of O(6)H  O(3) EH (kJ)

R (nm)

EH (kJ)

R (nm)

EH (kJ)

0.2766 0.2766 0.2766 0.2766 0.2767 0.2767 0.2767 0.2768

5.69 5.69 5.69 5.69 5.63 5.63 5.64 5.56

0.2786 0.2787 0.2787 0.2787 0.2787 0.2788 0.2789 0.2789

4.12 4.06 4.06 4.06 4.06 4.00 3.95 3.95

0.2812 0.2813 0.2814 0.2814 0.2814 0.2814 0.2814 0.2815

2.14 2.07 2.02 2.02 2.02 2.02 1.96 1.96

a/b

1.1 1.1 1.0 0.8 0.7 0.7 0.7 0.6

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Comparing the treated and untreated cellulosic samples, the appearance of both the amides I and II peaks is evident, and these are absent in the cotton cellulose spectra. Moreover, the increase of the intensity of these peaks with the increase in CBD concentration (from the samples B1 to B6, Fig. 7), and their shifting to a higher wavenumber, confirm the structural modification of cellulose structure after CBD binding. For further characterization, the peak at 2900 cm1 was selected as an internal standard, and the modification of cellulose structure during the adsorption process was established from the ratio between the intensity of the peak at 2900 cm1 and the intensity of the amide I peak in the range of 1700–1600 cm1 (Table 5). In addition, the FTIR absorption band at 1430 cm1, assigned to a symmetric CH2 bending vibration is shifted to a lower wavenumber during the CBD-binding process, and the intensity of this peak decreases with an increase in CBD concentration (Fig. 8). This band is also known as the ‘crystallinity band’, indicating that a decrease in its intensity reflects a decrease of the degree of crystallinity of the samples.22 This shifting, along with the fact that the band from 1316 cm1 that is assigned to CH2 wagging, moves toward 1317 cm1, indicates the development of new inter- and intramolecular hydrogen bonds.35,36 The FTIR absorption band at 898 cm1, which is assigned to C– O–C stretching at b-(1?4)-glycosidic linkages, and designated as an ‘amorphous’ absorption band, increases and moves to 902 cm 1 for B6 after treatment (Fig. 5). This fact additionally confirms a decrease in the degree of crystallinity of the samples, and an increase in the disordered regions.

Figure 8. FTIR absorption bands in the range of 1470–1260 cm1 for cotton cellulose (B) before and after treatment with CBD (samples B1–B6).

It is well defined that the ratios in absorbance of the FTIR bands, which are sensitive to the amount of crystalline versus amorphous structure in cellulose, CI1 = A1372/A2900 and CI2 = A1430/A899, are significant for the determination of the ‘crystallinity index’.23 As seen in Table 6, the CIs depend on the cellulose supramolecular structure and can be correlated with the degree of crystallinity determined by the X-ray diffraction method. In the case of the crystalline forms of cellulose samples, a decrease in the crystallinity follows the series B > L > C data, which are in good correlation with that obtained from XRD analyses. Also, it was shown that the CI values decrease with the increase of the CBD concentration due to the penetration of the CBD into the fibers and disruption of the hydrogen-bond network of the cellulose. The results obtained from the study of the ‘fingerprint region’ in the range of 1400–900 cm1 are in a good agreement with the changes observed in the broad band at 3600–3200 cm1, corresponding to the strong OH stretching and flexing vibration frequencies of intra- and intermolecular hydrogen bonds of cellulose. These reflect a structural transformation during the binding process of CBD onto cellulose fibers. 3.4. XPS studies

Figure 7. FTIR absorption bands in the range of 1700–1500 cm1 for cotton cellulose (B) before and after treatment with CBD (samples B1–B6).

Table 5 FTIR absorption bands in the range of 1700–1500 cm1 for cotton cellulose (B) before and after treatment with CBD (samples B1–B6) Sample Relative peak A1658/ Modif. (%) Relative peak A1548/ Modif. (%) area at 1700– A2900 area at 1600– A2900 1600 cm1 1500 cm1 B BK B1 B2 B3 B4 B5 B6

230.35 340.56 811.89 809.37 849.09 857.44 889.81 1024.6

0.42 0.56 0.83 0.86 1.05 1.02 1.10 1.22

— — 48.33 53.16 87.97 82.46 97.19 118.42

— — 16.62 17.54 24.37 33.12 37.02 35.38

— — 0.00 0.01 0.07 0.08 0.08 0.08

— — 0.00 10.00 43.00 45.64 48.41 48.22

In order to get information also about the surface chemistry of cellulose samples after treatment with CBD, XPS analyses were performed on selected samples. By applying the XPS method the elements present on the sample surface can be identified and

Table 6 The relative crystallinity index (CI) for cotton cellulose (B), linter cellulose (L), and dissolving pulp (C) before (initial) and after their treatment with different concentrations of CBD (1–6) and the corresponding blank samples Sample

Initial Blank 1 2 3 4 5 6

L—linter cellulose

C—dissolving pulp

CI1

B—cotton cellulose CI2

CI1

CI2

CI1

CI2

0.974 0.968 0.943 0.850 0.910 0.758 0.689 0.646

1.142 1.122 1.071 1.043 0.913 0.901 0.690 0.677

0.953 0.923 0.876 0.828 0.780 0.730 0.622 0.617

1.131 1.111 0.986 0.924 0.919 0.741 0.670 0.631

0.917 0.899 0.810 0.777 0.756 0.715 0.606 0.603

1.058 1.010 0.954 0.879 0.868 0.851 0.632 0.619

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Figure 9. XPS spectra from cotton cellulose (B) without and with CBD (samples B1 and B6).

quantified, and in addition, the chemical bonding of these elements can be recognized. Figure 9 shows the XPS survey spectra obtained for the untreated (B), and treated cellulose cotton samples with the lowest (B1) and the highest (B6) concentrations of CBD. The XPS spectrum of the untreated sample B shows only carbon C 1s and oxygen O 1s peaks, while for the CBD-treated samples nitrogen N 1s also appear. The analysis shows that the nitrogen concentration is larger for samples B6 (2.1 atom %) treated with higher concentrations of CBD compared with that for the lower CBD concentration applied to B1 (0.9 atom %). Other elements present in small concentrations such as K, P, and Na originate from the buffer solution, since they were detected also in the blank samples (BK). The surface concentrations of elements obtained from the XPS spectra are given in Table 7. The high-energy resolution XPS spectra were acquired in order to follow the chemical bonding present in the structure of untreated and treated cotton cellulose. The main oxygen peak O 1 is at 532.9 eV, which is related to different bonds such as C–O–C, C–OH, O–C–O, C@O, and a small peak also appear at

531.1 eV. The nitrogen N 1 peaks on the B1 and B6 samples are at 400.0 eV. For example, Figure 10 shows carbon C 1s and nitrogen N 1s spectra obtained on the sample B6. Detailed chemical-bond analysis of the carbon atoms was made by fitting the C 1s spectra by a least-squares method. Different components were recognized in the C 1s spectra: a peak C1 at 285.0 eV assigned to C–C/C–H bonds, a peak C2 at 286.3 eV assigned to C–O/C–OH bonds, and a peak C3 at 288.0 eV assigned to C@O/O–C–O bonds. Similar carbon C 1s spectra were obtained for other samples, but with different relative concentrations of C–C/C–H, C–O/C–OH, and C@O/O–C–O bonds. Table 8 shows relative concentrations of the C1, C2, and C3 peaks (carbon atoms differently bound with respect to all carbon atoms) recognized in the deconvoluted C 1s spectra and the ratio between some of these peaks. After the treatment with CBD, the peak C2 (C–O/C–OH) increased with the respect to C1, a fact which can be observed from the C2/(C1+C3) ratio (Table 8). According to other studies the C2 peak may be related to the fiber acidity, and the peaks C1 and C3 may be related to its basicity.37 Thus, after CBD adsorption the acidity of the fibers increased, which is in conflict with the data reported by Pinto et al.,3 probably because of the differences in the CBD protein amino acid structure and its folding properties on the cellulose surface, that is, its interactions with the cellulose surface.22 The relative increase of oxygen-related bonds with carbon atoms on the surfaces of the blank and treated samples is correlated with an increase of the O/C ratio, which can be the consequence of the unfolding of the CBD protein during the adsorption process, which turns hydrophobic amino acids to the interior of the cellulose surface and free carboxylic groups to the exterior interface, contributing to an increase of the fiber acidity. 3.5. SEM analysis Figure 11 presents SEM images, at different magnifications of untreated cellulose and those samples treated with the highest

Table 7 Surface concentrations in atom % of analyzed cellulose samples Sample

C (atom %)

O (atom %)

N (atom %)

K (atom %)

P (atom %)

Na (atom %)

B B1 B6

73.1 62.4 59.9

26.9 32.8 35.1

0.0 0.9 2.1

0.0 2.1 2.9

0.0 1.8 2.2

0.0 0.2 1.0

Figure 10. High-energy resolution XPS spectra (a) C 1s and (b) N 1s obtained on sample B6.

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D. Ciolacu et al. / Carbohydrate Research 345 (2010) 621–630 Table 8 Binding energy and relative concentrations obtained from XPS spectra of samples studied Element

XPS peak/binding energy

Carbon C 1s

C1: E = 285.0 eV C2: E = 286.5 eV C3: E = 288.0 eV O1: E = 531.1 eV O2: E = 532.8 eV

Oxygen O 1s O/C ratio N/C ratio C2/(C1+C3)

Relative concentrations (or area %) B

BK

B1

B6

53 ± 5 39 ± 4 8±2 1 ± 0.5 99 ± 10 0.37 0.00 0.64

47 ± 5 48 ± 5 4±2 14 ± 2 86 ± 9 0.59 0.00 0.94

41 ± 4 51 ± 5 8±2 25 ± 2 75 ± 8 0.53 0.01 1.03

26 ± 3 58 ± 6 16 ± 2 26 ± 3 74 ± 8 0.71 0.043 1.35

Figure 11. SEM images of (a) untreated cotton cellulose (B) and (b) treatment with CBD (B6).

concentrations of CBD. While a clear cellulose fiber surface can be seen in Figure 11a, in Figure 11b small particles appear spread over the cotton cellulose fibers, showing that the CBD adsorption implies a modification at the morphological level of the fibers, thus confirming the results obtained from XRD, FTIR, and XPS. It was shown that the CBD affects the supramolecular structure of cotton cellulose as the consequence of the disruption of the noncovalent binding between the cellulose chains. It seems that CBD can penetrate into the fibers and disrupt the weaker interactions between these, leading to a less ordered supramolecular structure of fibers.22 4. Conclusion The binding behavior of CBD from C. cellulovorans on cellulose fibers was studied using FTIR, XPS, XRD, and SEM analysis. FTIR spectroscopy was used to investigate the structural changes of cellulose that take place after the CBD adsorption process at the hydrogen-bond level. It appears that the intensity of the hydrogen bond decreased following the binding of CBD. The presence of bound CBD on cotton cellulose has been proved by the presence of characteristic amides I and II absorption bands in the FTIR spectra. A decrease in the crystallinity index during CBD adsorption was evidenced, indicating a disruption of intra- and intermolecular interactions, which was confirmed also by XRD measurements. Thus, it was proved that FTIR spectroscopy is a powerful tool to investigate the changes of cellulose supramolecular structure during CBD adsorption. From XRD it was shown also that CBD has different affinities for the different crystal faces of crystalline cellulose, with a preference for those in the (0 0 2) plane. The presence of CBD and its orientation on the cellulose surface was confirmed by XPS analysis, indicating an increase of the acid character of the cellulose after the CBD adsorption process. SEM analysis confirmed visible morphological changes on the cellulose

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